Multivalent binding of the hub protein LC8 at a newly discovered site in 53BP1.

Tumor suppressor p53 binding protein 1 (53BP1) is a scaffolding protein involved in poly-ADP ribose polymerase inhibitor hypersensitivity in BRCA1-negative cancers. 53BP1 plays a critical role in the DNA damage response and relies on its oligomerization to create foci that promote repair of DNA double-strand breaks. Previous work shows that mutation of either the oligomerization domain or the dynein light chain 8 (LC8)-binding sites of 53BP1 results in reduced accumulation of 53BP1 at double-strand breaks. Mutation of both abolishes focus formation almost completely. Here, we show that, contrary to current literature, 53BP1 contains three LC8-binding sites, all of which are conserved in mammals. Isothermal titration calorimetry measuring binding affinity of 53BP1 variants with LC8 shows that the third LC8-binding site has a high affinity and can bind LC8 in the absence of other sites. NMR titrations confirm that the third site binds LC8 even in variants that lack the other LC8-binding sites. The third site is the closest to the oligomerization domain of 53BP1, and its discovery would challenge our current understanding of the role of LC8 in 53BP1 function.

Continuum dynamics and statistical correction of compositional heterogeneity in multivalent IDP oligomers resolved by single-particle EM.

Multivalent intrinsically disordered protein (IDP) complexes are prevalent in biology and act in regulation of diverse processes, including transcription, signaling events, and the assembly and disassembly of complex macromolecular architectures. These systems pose significant challenges to structural investigation, due to continuum dynamics imparted by the IDP and compositional heterogeneity resulting from characteristic low-affinity interactions. Here, we developed a modular pipeline for automated single-particle electron microscopy (EM) distribution analysis of common but relatively understudied semi-ordered systems: 'beads-on-a-string' assemblies, composed of IDPs bound at multivalent sites to the ubiquitous ∼20 kDa cross-linking hub protein LC8. This approach quantifies conformational geometries and compositional heterogeneity on a single-particle basis, and statistically corrects spurious observations arising from random proximity of bound and unbound LC8. The statistical correction is generically applicable to oligomer characterization and not specific to our pipeline. Following validation, the approach was applied to the nuclear pore IDP Nup159 and the transcription factor ASCIZ. This analysis unveiled significant compositional and conformational diversity in both systems that could not be obtained from ensemble single particle EM class-averaging strategies, and new insights for exploring how these architectural properties might contribute to their physiological roles in supramolecular assembly and transcriptional regulation. We expect that this approach may be adopted to many other intrinsically disordered systems that have evaded traditional methods of structural characterization.

Human Parainfluenza Virus 3 Phosphoprotein Is a Tetramer and Shares Structural and Interaction Features with Ebola Phosphoprotein VP35.

The human parainfluenza virus 3 (HPIV3) poses a risk for pneumonia development in young children and immunocompromised patients. To investigate mechanisms of HPIV3 pathogenesis, we characterized the association state and host protein interactions of HPIV3 phosphoprotein (HPIV3 P), an indispensable viral polymerase cofactor. Sequence analysis and homology modeling predict that HPIV3 P possesses a long, disordered N-terminal tail (PTAIL) a coiled-coil multimerization domain (PMD), similar to the well-characterized paramyxovirus phosphoproteins from measles and Sendai viruses. Using a recombinantly expressed and purified construct of PMD and PTAIL, we show that HPIV3 P in solution is primarily an alpha-helical tetramer that is stable up to 60 °C. Pulldown and isothermal titration calorimetry experiments revealed that HPIV3 P binds the host hub protein LC8, and turbidity experiments demonstrated a new role for LC8 in increasing the solubility of HPIV3 P in the presence of crowding agents such as RNA. For comparison, we show that the multimerization domain of the Zaire Ebola virus phosphoprotein VP35 is also a tetramer and binds LC8 but with significantly higher affinity. Comparative analysis of the domain architecture of various virus phosphoproteins in the order Mononegavirales show multiple predicted and verified LC8 binding motifs, suggesting its prevalence and importance in regulating viral phosphoprotein structures. Our work provides evidence for LC8 binding to phosphoproteins with multiple association states, either tetrameric, as in the HPIV3 and Ebola phosphoproteins shown here, or dimeric as in rabies virus phosphoprotein. Taken together the data suggest that the association states of a virus-specific phosphoprotein and the complex formed by binding of the phosphoprotein to host LC8 are important regulators of viral function.

The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility.

Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest.